Abstract A core criterion of substance use disorder is continuing use of the rewarding substance despite clear consequences that would otherwise be highly aversive and thus behaviorally powerful in driving avoidance: harmful physical sequelae, negative social effects, and/or placement of the user in dangerous situations. Modern models of drug addiction invoke many possible sources of positive and negative reinforcement, but it is not known which specific circuitry is actually operative (causal) in allowing actions that cause normally-aversive physical harm, and it is not fully understood from the perspective of organismal-survival mechanisms how the destructive consequences of substance use could become entirely unable to deter drug use behavior. This remarkable conditionality of aversion, central to drug abuse, is also of fundamental significance in non- drug-related behavior; normally-aversive experiences can manifest with altered (neutral, or even positive) valence for a variety of adaptive and maladaptive reasons. In some cases, these effects might be understood by relatively simple neuroeconomic risk/benefit or gain/loss considerations; for example, sufficient reward (e.g. rare delivery of large food/water resources) may adaptively drive tolerance of same-class aversive events (e.g. temporary loss of food/water). We and others have studied and described these circuit computations extensively, including in the prior period of support from the present grant, by meticulous construction of carefully-balanced, defined-value, same-category reward/aversion stimuli. However, circuitry implementing this seemingly straightforward adaptive behavior (in which rewarding and aversive consequences are experienced, but expected-value of one is simply of greater magnitude) may be of insufficient complexity for the large majority of naturalistic situations, wherein reward and aversion are categorically different. In this proposal, we build upon our insights into casual, cell-specific reward circuitry, leveraging next-generation circuit-interrogation technology to identify (in brainwide fashion) the structurally- and molecularly-defined circuit elements at the intersection of reward and aversion, by which the vertebrate brain alters behavioral responses to aversive stimuli. In Aim 1, we develop next-gen CLARITY adapted to these paradigms to obtain brainwide wiring and molecular identification of all cells that are specifically recruited (active) in this key behavior (all registered to genetically-encoded Ca2+ sensor-derived activity data collected during the aversion- suppression behaviors). In Aim 2, using the tools from Aim 1 in combination with our latest optogenetic control methods, we will test specific circuit-activity hypotheses for causality in implementing cross-category modulation of aversion. In addition to the novel circuit targets that will emerge from the brainwide unbiased investigation of Aim 1, we already have specific circuit-level hypotheses to test based upon our existing preliminary data, ensuring that the later Aims of the proposal stand on an already-solid foundation. We hypothesize that this candidate circuit activity (in a tunable subset of projections from mPFC to specific subcortical structures) causes diminished behavioral impact of negative-valence stimuli via disrupted internal representation (and experience) of aversive stimuli. And in Aim 3, building on (and guided by) our identification of circuit elements that are naturally and causally involved in suppression of aversive responses, in this Aim we achieve single-cell real-time resolution during behavior. In the fiber-readout strategy of Aim 1 and 2; activity dynamics emerge as a single time-series from the fiber; as valuable as these data are, it is possible that signal increases could arise from altered synchrony or spread of activity in the region rather than from increased activity in a specific subset of target neurons. Single-cell resolution in causally implicated ensembles here will not only resolve this fundamental question, but also allow deep molecular profiling of the specific cells causally involved, with clear basic and translational implications. Identifying this circuitry will not only provide insight into the basic science of reward and aversion, but also will advance potentially revolutionary understanding and targeting of circuit elements that may be causal (or therapeutic) in human substance-use and neuropsychiatric disorders.